The chromogranin/secretogranins (or "granins') are a family of regulated secretory proteins found in the cores of amine and peptide hormone and neurotransmitter secretory vesicles. This family of proteins includes chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (Sgll). Evidence has now been gathered in support of both intracellular and extracellular functions for this protein family. Within the cells of origin, a granulogenic or sorting role in the regulated pathway of hormone or neurotransmitter secretion has been documented. Granins also function as pro-hormones, giving rise by proteolytic processing to peptide fragments for which activities have been demonstrated in vitro and in vivo. For instance, CgA fragments vasostatin and catestatin control vasoreactivity and catecholamine release, and the fragment pancreastatin elevates blood glucose. Prohormone processing mechanisms that generate active granin-derived peptides may involve the vesicular PC1 and PC2 prohormone convertases and the secretory granule cathepsin L. Using a series of novel granins chimeras, this project develop 4 specific aims directed to the understanding, in situ, of the trafficking and the storage of granins into catecholamine secretory granules, and to the comprehension of the dynamics of intravesicular pH and its role in the secretory process. In aim 1, we will use a series of CgA domains tagged with green fluorescent protein (GFP) or with embryonic alkaline phosphatase (EAP), to identify the sorting signals in CgA (cis determinant) that mediate chromaffin granule targeting of CgA. In aim 2, we will impair the biogenesis of chromaffin granules by silencing the expression of CgA, and use a series of CgA domains tagged with GFP or EAP to rescue or induce the formation of secretory granules, and identify CgA's granulogenic determinants. In aim 3, granin chimeras will be employed to investigate which features of the secretory apparatus (trans determinants) interact with CgA to influence its sorting, and its storage within the chromaffin granule. In aim 4, we wilt use pH-sensitive chimeric CgA photoproteins to investigate the dynamics of intravesicular pH and its rote in the secretory process triggered by the physiologic secretagogues or by sympathomimetic amines. The results of these studies will enhance our understanding of large dense-core secretory granule biogenesis, and of catecholaminelgranins storage and release during sympathetic stimulation.